Neutralized perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride copolymer surface for attachment and growth of animal cells

ABSTRACT

Neutralized surface of a polymer of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride for attachment and growth of animal cells in vivo or in vitro, the comonomer preferably being tetrfluorethylene.

FIELD OF THE INVENTION

This invention relates to the use of a copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomeras a surface for the attachment and growth of adherent animal cells. Theinvention has particular application to the manufacture and use ofprosthetic vascular grafts, connective tissue replacements and softtissue replacements that incorporate such a copolymer.

BACKGROUND ART

The design or selection of materials useful in vascular prosthesesrequires an understanding of the characteristics necessary forirreversible endothelialisation of a surface and for inhibition ofundesirable platelet interactions. An approach to the development ofvascular prostheses that has been taken has been guided by the object ofcircumventing the acute problems of platelet activation, adhesion andthrombogenesis. This approach involves designing a blood interface whichdisallows thrombogenesis by preventing platelet activation directly, andmay be achieved either by the selective incorporation or adsorption ofplatelet binding inhibitors, such as serum albumin or heparin, or byproviding a surface which directly repels or inactivates plateletselectrostatically. However these modifications might also suppress theattachment and growth of endothelial cells on the luminal surface of theprosthesis. Grafts prepared using this approach may therefore beregarded as unhealed and a physiological and anatomical state comparableto the normal luminal structure is not achieved.

It is generally known that surfaces which support endothelial cellgrowth comparable to that seen on glow discharged polystyrene also tendto be thrombogenic. However it is also known that sulphonatedpolystyrenes have antithrombogenic activity which is reported to be afeature of the negative charge of sulphonate groups. The presentinvention has been developed by following this line of investigation.

In a recent study, McAuslan and Johnson [(1987) J. Biomedical MaterialsResearch 21.921-935] showed that the hydroxyl rich surface ofpoly(hydroxyl ethyl methacrylate)(pHEMA) hydrogel can be converted froma non-cell adhesive to a highly cell adhesive state by either hydrolyticsurface etching or by copolymerization with methacrylic acid. Thus celladhesion appeared to correlate with the introduction of surface COOHgroups although this alone was not a sufficient condition. This hasraised the question of whether other negatively charged moieties wouldbe just as effective at promoting cell attachment.

A fluorocarbon polymer with pendant sulphonic groups is the chemicallyinert, non-crosslinked cation-exchange resin known by the trade markNAFION. NAFION is chemically identified as a copolymer oftetrafluoroethylene and perfluoro-3,6-dioxa-4-methyl-7-octene sulphonylfluoride. The mechanical and chemical stability of thisperfluorosulphonate ionomer and its selective permeability to chargedions had made it useful for industrial electrochemical separatingprocesses. It can be prepared as films or tubes and is hydrophilic,which is in contrast to polytetrafluoroethylene (PTFE, which is known bythe trade mark TEFLON) or expanded PTFE (which is known by the trademark GORE-TEX), a material which is in wide use as a vascular graft.

We have now found that any copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomer,and particularly NAFION, may, when in a neutralised form, be used as asurface for the attachment and growth of adherent animal cells fromdifferent tissue sources, including endothelial cells. In thisspecification and claims, reference to being in a neutralised form meanswithin one pH unit of pH 7.0.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a material useful invascular prostheses and other implantables having improvedbiocompatibility arising from enhanced endothelial cell attachmentproperties and anti-thrombogenicity which will substantially overcomethe disadvantages of the prior art.

In accordance with one aspect of the present invention, there isprovided a surface for the attachment and growth of cells in vivo, saidsurface comprising the neutralized form of a copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomer.Preferably, the monomer is tetrafluoroethylene. The above surface may beadsorbed or attached to an appropriate substrate that is preferablyporous. The types of substrate that may be used include polymers,ceramics, metals, glass or preformed membranes. When a polymer substrateis used, the polymer is preferably porous such aspolytetrafluoroethylene, expanded polytetrafluoroethylene, knitted orwoven polyester and polyurethane.

In a preferred form, the surfaces of the present invention that may beused in vivo are in the form of a sponge or tube and may be adapted foruse in a biosensor. The surfaces of the present invention that may beused in vivo may also be modified by selective incorporation of plateletbinding inhibitors such as serum albumen or heparin or by treating thesurfaces with agents that specifically repel or inactivate plateletattachment.

In accordance with another aspect of the present invention, there isprovided a surface for the attachment and growth of cells in vivo, saidsurface comprising the neutralised form of a copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomeroptionally adsorbed or attached to an appropriate substrate as describedabove, and wherein said surface further includes adsorbed adhesiveproteins. The preferred adhesive proteins are derived from serum andinclude fibronectin, vitronectin or adhesive fragments of theseproteins. Other preferred adhesive proteins include laminin, collagensand thrombospondin and adhesive fragments of these proteins. The abovesurfaces that may be used in vivo, may also have adsorbed theretoadhesive proteins or their adhesive fragments.

In a further preferred embodiment, the above surfaces for use both invivo and in vitro include adhered cells of the type sought to be grown.

In accordance with yet another aspect of the present invention, there isprovided a process for the preparation of a surface for the attachmentand growth of cells in vivo, said process comprising applying acopolymer of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluorideand a monomer to an appropriate substrate. The surface so prepared mustbe brought to neutrality, and this may be done by either neutralizingthe resultant surface or by applying the above copolymer in aneutralized form to the appropriate substrate.

The above copolymer is preferably applied to the substrate by means ofradiation grafting or adhesive bonding.

In accordance with a further aspect of the present invention, there isprovided a process for the preparation of a surface for the attachmentand growth of cells in vitro, said process comprising exposing adhesiveproteins or adhesive serum proteins to a surface comprising theneutralized form of a copolymer of perfluoro-3,6-dioxa-4-methyl-7-octenesulphonyl fluoride and a monomer optionally adsorbed or attached to anappropriate substrate, whereupon the copolymer adsorbs said proteins toform a copolymer-protein complex.

In a further preferred embodiment, the surfaces prepared according tothe above processes for use both in vivo and in vitro are furtherexposed to cells of the type sought to be grown, whereupon the cellsadhere to the said surface to further improve its cell attachment andgrowth properties.

In accordance with a still further aspect of the present invention,there is provided a method for the attachment and growth of cells to asurface both in vivo and in vitro, which method comprises exposing cellsor a medium containing cells and adhesive proteins to a surface of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the relative growth rates of bovine aortalendothelial cells on the following surfaces: tissue culture plastic(TCP), NAFION 22, 70 and 125 films prepared as described in Example 1,during the period of 4 days after seeding, and,

FIG. 2 (A and B) shows the difference in cell morphology and celldensity of bovine aortal endothelial cells after 4 days culture onuntreated TEFLON (FIG. 2A) or on TEFLON that was coated with NAFION, asdescribed in Example 1, (FIG. 2B). Cells were stained with acridineorange and photographed under UV light and the photographs are at 80times magnification.

FIG. 3a is a graph of the relative growth rate of the human cell lineHep-2 on the following surfaces: tissue culture plastic, NAFION 70prepared as described in Examples 1 and 3 and both the above surfacesprecoated with fibronectin prior to cell seeding, cultured for a periodof 7 days.

FIG. 3b is a graph of the relative growth rate of the human cell lineHeLa on the following surfaces: tissue culture plastic, NAFION 70prepared as described in Examples 1 and 3 and both the above surfacesprecoated with fibronectin prior to cell seeding, cultured for a periodof 8 days.

FIG. 3c is a graph of the relative growth rate of the human cell line HT1080 on the following surfaces: tissue culture plastic, NAFION 70prepared as described in Examples 1 and 3 and both the above surfacesprecoated with fibronectin prior to cell seeding, cultured for a periodof 7 days.

FIG. 4 (A-D) shows the difference in morphology of human umbilicalarterial endothelial cells cultured on tissue culture plastic (FIG. 4Aand FIG. 4C) or NAFION 70, prepared as described in Examples 1 and 3(FIG. 4B and FIG. 4D). Some of the surfaces (FIG. 4C and FIG. 4D) wereprecoated with fibronectin prior to cell seeding. The cells werecultured for 7 days then photographed. The photographs in FIG. 4A andFIG. 4B are at 110 times magnification and in FIG. 4C and FIG. 4D, at220 times magnification. Note that the morphology of the cells on NAFION70 is indistinguishable from that of the cells on the tissue cultureplastic, and not also the enhanced cell spreading on each of thesurfaces when precoated with fibronectin.

FIG. 5a is a graph of the relative growth rates of the human umbilicalarterial endothelial cells cultured for 7 days on tissue culture plastic(TCP) and TCP precoated with fibronectin prior to cell seeding.

FIG. 5b is a graph of the relative growth rates of the human umbilicalarterial endothelial cells cultured for 7 days on TEFLON that was coatedwith NAFION (NAF) as described in Example 1, and NAF precoated withfibronectin prior to cell seeding.

FIG. 5c is a graph of the relative growth rates of the human umbilicalarterial endothelial cells cultured for 7 days on untreated TEFLON(TEF), and TEF precoated with fibronectin prior to cell seeding.

FIG. 6 (A-F) shows the difference in morphology of human umbilicalarterial endothelial cells cultured on GORE-TEX (FIG. 6A and FIG. 6B),or GORE-TEX that was coated with NAFION as described in Example 3 (FIG.6C and FIG. 6D). Another sample of NAFION-coated GORE-TEX (FIG. 6E andFIG. 6F) was precoated with fibronectin prior to cell seeding. The cellattachment and morphology was examined by scanning electron microscopyand the magnification of each panel is given as follows: for FIG. 6A,FIG. 6C and FIG. 6E, 200 microns=62 mm on the print, whereas for FIG.6B, FIG. 6D and FIG. 6F, 50 microns=61 mm on the print. Note the sparcecell attachment to GORE-TEX (FIG. 6A and FIG. 6B) but markedly bettercell coverage and spreading on the GORE-TEX that was coated with NAFION,giving almost complete cell coverage of the surface (FIG. 6C to FIG.6F). The cells were fixed after 1 day of culture (FIGS. 6A, 6B, 6C, 6D,6F) or 3 days of culture (FIG. 6E).

FIG. 7 is a diagram of the apparatus used to test cellular attachmentunder conditions of flowing culture medium, as described in Example 4.

DESCRIPTION OF PREFERRED EMBODIMENTS

In order that the invention may be more readily understood and put intopractical effect, reference will now be made to the following examplesthat describe the use of NAFION as a preferred embodiment of thecopolymer of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluorideand a monomer.

EXAMPLE 1 Preparation of NAFION N117, NAFION N125, NAFION 22 and NAFION70 membrane preparations, and PTFE and GORE-TEX coated with NAFION 70.

Portions of NAFION N117 membrane and NAFION N125 membrane were cut into1 cm² pieces and washed in acetone followed by absolute ethanol. Thepieces were then treated with 0.2% EDTA to remove cationic contaminants,washed thoroughly with deionised water and sterilised by autoclaving.Prior to tissue culture studies the pieces were extensively washed insterile phosphate buffered saline (PBS) pH 7.2.

A 5% (w/vol) solution of NAFION 1100 Equivalent Weight perfluorinatedion-exchange resin was used for casting the following membranes:

i) NAFION 22--prepared by casting 0.5 ml 5% NAFION solution in the lidof a 35 mm diameter tissue culture petri dish as 22° C.

ii) NAFION 70--prepared by repeating the procedure for NAFION 22 at 70°C. for 2 hours.

Prior to tissue culture studies, the NAFION 22 and NAFION 70 membraneswere sterilised under ultraviolet light for 2 hours and then extensivelywashed in serum free tissue culture medium.

iii) Unfilled virgin TEFLON samples were washed extensively in ethanoland some were coated with NAFION solution and some were left uncoated,for cell culture studies. Approximately 1 cm² pieces of material werecoated with 50 microliters of 5% NAFION Equivalent Weight 1100 solutionand treated at 70° C. for 2 hours. Substrates so prepared and tissueculture polystyrene (TCP) were sterilised under UV light for 2 hours andwashed extensively in serum free tissue culture medium before being usedfor cell culture studies.

Equilibrium water content (EWC)

The EWC of both NAFION N125 and the NAFION 22 and 70 membranes wasdetermined essentially as described by Pedley and Tighe ](1979) Br.Polym. J., 11, 130-135]. In both cases the NAFION was pretreated with0.2% EDTA and then washed and equilibrated in deionised water for 4 to 6days before weighing.

Results

Commercially available preformed sheet NAFION (NAFION N-117 and N-125)and NAFION membrane cast from a 5% solution on either glow discharged ornon-glow discharged polystyrene at 22° C. (NAFION 22) were transparentneutral coloured substrates. After casting at 70° C. (NAFION 70), nodifference in texture of colour was observed. However this treatmentrendered it insoluble in ethanol and acetone. NAFION membranes were castin a variety of thickness from approximately 10 to 40 microns. Bycomparison the preformed sheet NAFION used in this study wasapproximately 100 microns thick. NAFION N125 has an equilibrium watercontent of 12.0% and NAFION 22 and 70 equilibrium water contents of 32%and 36% respectively. NAFION membranes cast on polystyrene could bepeeled off the surface by gently pulling with forceps and the thicknessof the membrane determined the fragility of such material.

EXAMPLE 2 Attachment and Growth of Bovine Endothelial Cells and otherAdherent Animal Cells on NAFION Methods Cell Culture and Cell GrowthRate Determination

A clonal line of normal bovine aortic endothelial (BAE) cells were grownand maintained in M199 cell culture medium supplemented with 20% (v/v)fetal calf serum. To determine the increase in the number of cellsgrowth on the different substrates, 2 ml of M199 cell culture mediumsupplemented with 20% foetal bovine serum containing between 5×10⁴ and2×10⁵ BAE cells were added to dishes containing NAFION N125, or coatedwith either NAFION 22 or NAFION 70, and incubated in a humidifiedatmosphere of 5% CO₂ in air at 37° C. After 6 hours, cell attachment wasestimated by counting cells within 15 randomly chosen 0.931 mm² areas.Each polymer sample was the subject of three individual trials and meancell numbers expressed per cm². After this initial period cell numberswere determined every 24 hours to determine cell growth rate. Forcomparison the growth of BAE cells on GORE-TEX, TEFLON and glowdischarged tissue culture polystyrene was also determined. Cellmorphology was investigated by routine light microscopy of culturedsurfaces for the NAFION membranes. For the NAFION cast onto TEFLONsurfaces, the opacity of the TEFLON precluded the use of phase contrastmicroscopy for a full visual comparison, so cells were fixed with 2.5%gluteraldehyde, stained with 3 mM acridine orange then photographedunder UV light.

Results

It is known that NAFION is a strong acid and washing the cast polymers(NAFION 22 and NAFION 70) in serum-free tissue culture medium showedthat a comparatively large volume of medium was required to neutraliseits acidic property. This is therefore, an important aspect of the useof NAFION as a substrate for cell growth which must be taken intoaccount in its preparation. Similarly, NAFION preformed membrane (NAFIONN117 and NAFION N125) required neutralisation before use for cellculture.

A wide range of different cell types have been successfully grown onNAFION. These include bovine aortic endothelial cells (BAE), bovineaortic smooth muscle cells, bovine corneal endothelial cells, bovineretinal capillary endothelial cells, baby hamster kidney fibroblasts,and 3T3 fibroblasts. All cell types displayed their own characteristicmorphology and growth characteristics when observed on TCP controldishes. Endothelial cells formed a cobblestone pavement monolayer whilstfibroblasts showed spindle shaped morphology and eventually formed awhorl-like pattern.

Since it was proposed that the material might show potential as acomponent of a vascular prosthesis the attachment and growth of BAEcells on NAFION compared to TCP and TEFLON was studied in some detail.Cell attachment in vitro was found to be dependent on the presence ofserum; and in the absence of serum no cells were attached to any of theNAFION preparations after six hours. The attachment of BAE cells to thedifferent substrates after 6 hours was expressed with respect to cellattachment to tissue culture polystyrene (which was set as 100%). TheBAE cell attachment to NAFION 70 and NAFION 22 was 109±3.2% (MeanStandard Deviation) and 97±3.6% respectively; to NAFION N125, 84±3.2%and to TEFLON, 75±6.8%. Of particular interest is the fact that cellsappeared to be more evenly distributed on the surface of the NAFIONfilms that on NAFION N-125 or tissue culture polystyrene. FIG. 1 showsthe kinetics of growth of BAE cells on NAFION N-125, 22 and 70 comparedto their growth on tissue culture polystyrene after 4 days. Only smalldifferences were seen in the growth rates and final numbers of cells onthe NAFION preparations compared with TCP. The growth rate of BAE cellson NAFION preparations observed in these experiments was considerablyhigher than the level recognised for such cells on the commonly usedTEFLON vascular graft material. Cell growth on NAFION coated TEFLONshowed a similar improvement, over such cells grown on TEFLON alone. Bycasual appraisal cells cultured on TEFLON had a slower growth rate thancells cultured on the other polymers. In contrast to BAE cells seeded onNAFION, cells seeded on TEFLON failed to achieve the characteristicpolyhedral morphology and remained fibroblastoid until almost confluent.This effect was demonstrated by growing 10⁵ BAE cells/ml in culture for4 days on untreated TEFLON and NAFION coated TEFLON. BAE cells grown onuntreated TEFLON displayed a typical patchiness or fibroblast-likemorphology in the areas of sparse cover (FIG. 2a). This ischaracteristic of BAE cells when growing on a less than ideal substrate.In contrast, BAE cells grown on NAFION coated TEFLON achieved thepolyhedral morphology characteristic of such cells grown on idealsubstrates (FIG. 2b). Further, BAE cells could be maintained in cultureon NAFION N125 successfully for up to 3 weeks.

The growth of BHK fibroblasts on non-glow discharged polystyrene wasfacilitated by coating the surface with NAFION solution. In accordancewith the findings for BAE cells, no difference between the behaviour ofBHK fibroblasts to NAFION 22 compared to glow discharged polystyrene wasseen whereas cells failed to attach and spread properly or nonglowdischarged polystyrene (results not shown). Attachment of BAE cells toTEFLON was less strong than to polystyrene or NAFION as seen when onlygently pipetting of medium or PBS with a Pasteur pipette was sufficientto detach the cells from the TEFLON surface. Such physically weakattachment to tissue culture polystyrene and NAFION was not seen.

EXAMPLE 3 Attachment and Growth of Human Endothelial Cells and otherHuman Cells on NAFION 70 Membrane Preparations. Methods

In some experiments of this Example, the serum adhesive glycoproteinfibronectin (Fn) was removed from serum prior to use of the serum forcell culture by passage over a gelatin-Sepharose affinity column. Serumtreated on a gelatin-Sepharose column was confirmed to be free of Fn byimmunoassay of the Fn content. In other experiments, the serum adhesiveglycoprotein vitronectin (Vn) was removed by passage over an affinitycolumn consisting of immobilized anti-Vn antibody. The sera that weredepleted in Vn by this affinity technique were confirmed to have beenexhaustively stripped of vitronectin by immunoassay for Vn content.

Human cell lines HeLa from cervical carcinoma, HeP-2 from carcinoma ofLarynx, and HT 1080 from human fibrosarcoma were grown in a growthmedium consisting of minimal essential medium supplemented with 10%(v/v) foetal calf serum, 60 microgram/ml penicillin and 100 microgram/mlstreptomycin.

A human umbilical artery endothelial (HUAE) cell culture was establishedand grown in 75 cm² tissue culture polystyrene (TCP) flasks coated withFn. Coating with Fn was achieved by incubating the flasks with 5 mlsolution of 40 ug/ml Fn in PBS at 37° C. for 1 hour prior to cellseeding. Excess solution was removed before cells were added. The cellswere routinely maintained in a growth medium consisting of an equalmixture of McCoy 5A (modified) and BM86-Wissler media supplemented with30% v/v foetal bovine serum, 40 ng/ml fibroblast growth factor, 60 ug/mlendothelial cell growth supplement, 20 ug/ml insulin, 60 ug/mlpenicillin and 100 ug/ml streptomycin. The cells were routinely passagedusing trypsin-versene, and for experimental work cells were used betweenpassage 15 and passage 20 (inclusive).

For attachment and cell growth studies, NAFION 70 films cast onto 22 mlwells were equilibrated with PBS, and 2 ml of growth medium containing5×10⁴ cells was added to each well. The cell attachment was determinedby counting, in randomly selected field, the total number of cells andthe number of these cells that had spread onto the surface. Cell growthin each well was quantitated by counting 5 randomly selected fields perwell after successive days of culture, until cell confluence wasreached. The mean and standard error of cell number per cm² fortriplicate samples were determined.

GORE-TEX samples were cut into pieces of approximately 1 cm², coatedwith approximately 0.3 ml NAFION solution per piece, then immediatelytreated at 70° C. for 2 hrs. The NAFION-coated GORE-TEX was exposed toUV light for 2 hrs and then washed extensively in serum-free tissueculture medium. Some samples were then coated with 40 ug/ml Fibronectinfor 45-60 min at 37° C. prior to seeding with HUAE cells.

Results

The attachment and growth of human cell lines Hep-2, HeLa and HT 1080was compared to that on tissue culture polystyrene (TCP). Cellattachment and growth was also determined on NAFION films that had beencoated with a solution of 40 ug/ml Fibronectin. The human cell linesHep-2, HeLa and HT 1080 all attached and grew on NAFION films (see FIG.3 for cell growth curves). It was necessary to use culture medium thatcontained serum for the cells to attach and to grow on the NAFIONsurface. In the case of cell lines Hep-2 and HeLa, the rate of cellgrowth was increased where the NAFION film had been precoated with Fn(see FIG. 3 for comparison of Fn coated NAFION and NAFION that had notbeen coated with Fn) whereas in the case of HT 1080 cells the Fn coatingof the NAFION had no positive effect on the cell growth rate.

HUAE cells were grown on NAFION and compared to growth on TCP. It wasalso necessary to use culture medium that contained serum for the HUAEcells to become attached and to grow. Fn-coated TCP was also included asa control surface, as this surface is known to support good HUAE cellattachment and growth. The number of HUAE cells attached to the NAFIONsurface as viewed after 4 hours fo cell seeding was equivalent to thaton TCP, whereas for the Fn-coated NAFION, the number of cells attachedwas equivalent to that on the Fn-coated TCP. The morphology of the HUAEcells attached to the NAFION surface was generally similar to that ofthe HUAE cells seeded onto TCP, see FIG. 4. This morphology indicatedthat although the HUAE cells had attached to the NAFION surface whenseeded in the presence of serum, the cells had not formed the wellspread morphology that is typical of HUAE cells that have been seededonto Fn-coated TCP. However the morphology of the HUAE cells thatattached to the Fn-coated NAFION films was well spread and the cellmorphology was similar to HUAE cells growing of Fn-coated TCP, see FIG.4.

HUAE cells grew on the NAFION and Fn-coated NAFION surfaces at a ratethat was similar to the cell growth on TCP and Fn-coated TCP,respectively (see FIG. 5 for growth curve).

The role that Fn from the serum and Vitronectin from the serum may playin the attachment of the HUAE cells to the NAFION and Fn-coated NAFIONsurfaces was determined by selective removal of these components fromthe serum used in the culture medium in which the cells were seeded.Selective removal of Vn from the culture medium completely abolished theattachment of HUAE cells to the NAFION surface. The importance ofVitronectin (which is also known as serum spreading factor, epibolin or70K spreading factor) has been previously reported for other polymersurfaces such as tissue culture polystyrene, see Grinnell [(1976) Exp.Cell Res., 97, 265-274 and (1977) Exp. Cell Res., 110, 175-190].Attachment of HUAE cells to Fn-coated NAFION over a 4 hour period whenseeded in culture medium containing Vn-depleted serum was equivalent tothat of HUAE cells seeded in intact medium onto the Fn-coated surface.

The selective removal of Fn from the seeding culture medium did notabolish the attachment of HUAE cells to the NAFION surface. As aconsequence of removal of serum Fn, the rate of cell attachment wassomewhat reduced over the first 4 hours as compared to cell attachmentto NAFION where the culture medium contained intact serum. However after24 hours the HUAE cell coverage of the NAFION surface with theFn-depleted culture medium was identical to that seen on the NAFIONsurface with the medium containing intact serum. The use of culturemedium containing vitronectin-depleted serum for seeding of HUAE cellsonto Fn-coated NAFION surface did not effect the rate and extent of HUAEcell attachment and cell morphology, as compared to that where the HUAEcells were seeded onto Fn-coated NAFION using culture medium containingintact serum.

These results indicate that the serum-dependence of the attachment, cellspreading and growth of HUAE cells on a NAFION surface that has not beenprecoated with purified Fn or other adhesive proteins involves as anessential component the serum adhesive glycoprotein Vitronectin.Vitronectin from serum or culture medium containing serum is known fromprevious work to adsorb readily onto other culture surfaces such astissue culture polystyrene. The results also indicate that the NAFIONsurface is similar to other surfaces used for the attachment of cells inthat the adhesive glycoprotein Fn may be purified from serum and thencoated onto the NAFION surface to give a substratum that supports goodHUAE cell attachment, with consequential effects on cell growth. Takentogether, these results indicate that the adsorption of adhesive serumproteins such as vitronectin or fibronectin onto a NAFION surfaceproduces a substratum for promoting cell adhesion and growth.

GORE-TEX-NAFION-Fn as a substratum for HUAE cell growth

NAFION was coated onto GORE-TEX, then the NAFION GORE-TEX surface wasseeded with HUAE cells. In some samples the NAFION-GORE-TEX surface wasprecoated with Fibronectin prior to cell seeding. The HUAE cellsattached to the NAFION-GORE-TEX surface and grew to produce a surfacethat was almost completely covered with HUAE cells (See FIG. 6). TheHUAE cells grown on each of the NAFION-GORE-TEX surface and thefibronectin-coated nation-GORE-TEX surface had a well attached andspread morphology as observed in the scanning electron microscope (seeFIG. 6).

EXAMPLE 4 Attachment and Growth of Ovine Endothelial Cells on NAFIONtubes. Methods

An ovine carotid arterial endothelial (OCAE) cell culture wasestablished after the methodology of Jaffe ((ed) Biology of EndothelialCells (Developments in Cardiovascular Medicine) 1984, Martinus NijhoffPublishers, Boston), and routinely maintained in McCoy 5A (modified)medium supplemented with 20% foetal bovine serum, 60 ug/ml penicillinand 100 ug/ml streptomycin and passaged using trypsin-versene. Forexperimental work, cells were used between passage 6 and passage 12(inclusive). Preequilibrated NAFION tubes (2.9 mm internal diameter and25 mm in length) were incubated with a 40 ug/ml solution of fibronectin(Fn), washed with PBS and individually placed into sterile-cappolystyrene vials, then 9 ml of growth medium containing 2×10⁶ cells wasadded to each via. The cell suspension was gassed with a mixture of 5%CO₂ in air and the vial tightly sealed. The vials were then placedinside a TCP roller bottle and firmly held in a position by packing. Theloaded bottle was then rotated at 1 r.p.m. on a roller at 37° C. Theculture medium was replenished at 24 hr and 72 hr and the tubes removedfor subsequent flow testing after 5 days. Cell growth could be observedthrough the NAFION tube using a phase contrast microscope. Havingobserved that the tubes support cell attachment and growth over 5 days,the tubes were cultured for 6 hr in culture medium consisting ofDulbecco's modified Eagle's medium containing glutamine, 3 mg/lmethionine and 25 uCi/ml of 35S-methionine, then further incubated withthe normal (McCoy 5A medium with serum and supplements) medium for afurther 15h. The tubes containing the metabolically-labelled cells werebriefly washed in PBS then inserted into the flow test system asdetailed in FIG. 7. The tubes were subjected to increasing flow rates ofa medium consisting of McCoys 5A medium containing 20 mM Hepes buffer(pH 7.2) and 20% (v/v) foetal bovine serum at 37° C. for the specifiedtime periods. Cells released from the tubes were collected on thedownstream glass fibre filters and quantitated by radioactivedetermination (liquid scintillation counting). Following the flowstudies, the tube was removed and bisected then half of the tube wasexamined for adherent cells by microscopic techniques and the cells onthe other half were removed using trypsin-versene and the radioactivityin the released cells was determined

Results

In view of the results with HUAE cells where enhanced cell spreading,attachment and growth was produced by precoating the NAFION surface withFibronectin (Example 3 above), the NAFION tubes that were used in theflow experiments were precoated with Fn. OCAE cells seeded into theFn-coated NAFION tubes attached to the luminal surface and formed aconfluent monolayer of cells during 3 to 5 days of culture. The cellsattached to the tube were tested for cellular attachment in an in vitroflow system that permitted laminar flow (at flow rates up to 207 ml/min,equivalent to 12.6 dynes/cm² shear force, and above this flow rate,turbulent flow, see FIG. 7 for design). The OCEA cells withstood theshear force treatments of up to 20 dynes/cm², with negligible celldetachment during the flow treatment (see Table 1 below). The cellularmonolayer was examined microscopically after the flow treatment and thecells remained attached and well spread to the luminal surface of thetube, with no evidence of detachment or damage to the cells. Theseexperiments show that the endothelial cells form strong attachment tothe Fn-coated NAFION tube surface and can withstand shear forces thatare equivalent to those that would be encountered in vivo.

                  TABLE 1                                                         ______________________________________                                        Retention of OCAE cells on NAFION tubes under                                 fluid flow conditions                                                                    % cells remaining                                                                          % cells detached and                                  Experiment attached to tube                                                                           recovered on filters                                  ______________________________________                                        1          99.4         0.6                                                   2          99.1         0.9                                                   3          99.0         1.0                                                   4          98.2         1.8                                                   5          97.8         2.2                                                   ______________________________________                                    

The cells were cultured on Fibronectin-coated NAFION tubes as describedin the text above, then the cells attached to the tubes were subjectedto the following flow protocol: 10 min at a flowrate of 66 ml/mincorresponding to a shear force of 4 dynes/cm² followed by 10 min at 132ml/min equivalent to 8 dynes/cm², then 10 min at 198 ml/min equivalentto 12 dynes/cm², then 10 min at 264 ml/min equivalent to 16 dynes/cm²,then 10 min at 330 ml/min equivalent to 20 dynes/cm². It should be notedthat the increase in flow corresponding to the step going from 12 to 16dynes/cm² necessitated going from laminar to turbulent flow.

EXAMPLE 5 Studies of in vitro thrombogenicity of NAFION surfaces.

In vitro thrombogenesis of TEFLON, NAFION, tissue culture polystyreneand vitrogen coated polystyrene surfaces, were studied in the followingmanner:

(i) Human Platelet Binding

Human platelets prepared from fresh human plasma were labelled withChromium 51. Platelets collected by centrifugation were labelled withCr-51 in 0.25 M HEPES/tris buffer pH 7.0 containing a stock solution ofCr-51 in 0.2 HEPES/tris buffer for 1 hr at room temperature. Thepercentage incorporation of Cr-51 into 10 platelets was checked bycounting the amount of radioactivity incorporated in platelets collectedby centrifugation and unincorporated radioactivity in the supernatent.Incorporation of the label was then inhibited by the addition of 5%ascorbic acid. Platelets were incubated in the presence of the polymersurface under study in a 96 well ELISA tray for 3 hours at roomtemperature. The polymers were the removed and washed thoroughly in0.14M NaCl/0.02 HEPES buffer pH 7.0 and bound radioactivity determinedby counting the polymers in a gamma counter. Results were expressed asnumbers of platelets bound to the polymer surface per mm².

(ii) Partial thromboplastin time

Partial thromboplastin time was determined by incubating platelet poorplasma (Prepared by centrifugation) in the polymer surface in glasstubes. 0.1M NaCl was added to the plasma and the time for clot formationmeasured. This time was taken as the partial thromboplastin time.

Results

The platelet binding experiments showed that polystyrene, TEFLON andNAFION bound between approximately 12,000 and 18,000 platelets per mm²whereas polystyrene coated with vitrogen 100 (purified collagen) bound163,682 platelets per mm². Actual figures obtained were for polystyrene15,023; for TEFLON 17,860 and for NAFION 18,070 platelets per mm².Partial thromboplastin times revealed that NAFION required 210 secondsfor clot formation followed by vitrogen 100-coated polystyrene thatrequired 238s; TEFLON 247 s and polystyrene 250 s.

These data indicate that NAFION and TEFLON have similar partialthromboplastin times and platelet binding properties (low, compared tocollagen surfaces) which suggests that NAFION is no more thrombogenicthan TEFLON.

EXAMPLE 6 Porous NAFION Implants

The use of autograft material is still the most desirable method used toreplace diseased or damaged tissues or organs. However, due toanatomical or other considerations, such as those of infection orrejection, this approach may not be feasible. Of particular challenge isthe repair of connective tissue defects. Both naturally derived andsynthetic materials have been used in this regard, for example,injectable solubilized collagen and polymeric hydrogels. There isincreasing interest in the provision of synthetic materials ascomponents of prosthetic devices.

Methods

NAFION implants were prepared by mixing a 5% solution of NAFIONEquivalent Weight 1100 with NaCl crystals in approximately 10:1 ratio.The mixture was poured into either glass petri dishes or small (20 ml)volume beakers and incubated at 60° C. for between four and seven days.After this time the NaCl was dissolved in distilled water. However,implants could have been made by alternative techniques described in theart, e.g. sintering, thermal expansion, laser or ion beam drilling, etc.The material samples were sterilized prior to implantation using anindustrial method of ethylene oxide processing. In vivo biocompatibilitytesting was conducted using males of an inbred strain of BALB/c mice.Animals were anaesthetised using ether and their dorsal and flankregions prepared by clipping the fur and swabbing with HIBITANEdisinfectant (10% in 70% ethanol, each (v/v)). Implants were insertedsubdermally by making a small incision with a pair of scissors andfurther blunt dissection to prepare a small pocket into which eachimplant was placed. The skin was closed using two sutures (Mersilk 4.0)and swabbed again with HIBITANE. Each animal received one implant of thematerial and was caged separately after surgery. Animals were biopsiedafter one, three, four, six and twelve weeks. At biopsy, implants wereexamined macroscopically for signs of lysis or gross inflammation,excised with the overlying skin and fixed in 10% formaldehyde in normalsaline. The tissue was dehydrated in ethanol and prepared for routinehistology by embedding in Historesin (LKB); 2 mm sections were stainedwith haematoxylin and eosin and viewed in an Olympus BH-2 microscope.

Results

All implants were recovered progressively up to 12 weeks. Macroscopicexamination revealed that no overt tissue inflammation was present. Anindication of the resistance of the implant to cellular degradation wasthat the original angular contours of the cut edges of the implants werestill visible even after 12 weeks in vivo. Implants appeared to maintaintheir original size throughout the 12-week study period. Histologicalexamination showed that after one week, implants were infiltrated withlymphocytes and a highly cellular connective tissue capsule had formedaround the periphery. Lymphocytes had also migrated into the centre ofthe implants. Three and four week implants appeared similar. Peripheralaspects of the implants were well embedded in the fibrous tissuesurrounding the implant. Some fibrous tissue ingrowth was seenparticularly after four weeks, as was the presence of large capillaries.At this stage multinuclear giant cells were seen in close associationwith the implants. After six weeks cellular and fibrous tissue ingrowthwas well developed. The lymphocyte inflammatory response was reduced bythis time however more multinuclear giant cells were found around theedges of the implants. Twelve weeks after implantation, isolated fingersof fibrous tissue had extended into the centre of most of the implants.These were populated by fibroblasts and lymphocytes and contained smallblood capillaries. All implants were well embedded in host tissue andlarger blood vessels were observed growing across the surface of theimplants. The implants showed no sign of material degradation.

In summary Examples 1 to 5 demonstrate the excellent cell response toNAFION in different forms. By successfully growing a number of mammaliancell types, notably human arterial endothelial cells, on a variety ofNAFION substrates we have shown that NAFION has good cell supportivecharacteristics. In particular, we have demonstrated the efficacy ofNAFION coated TEFLON for support of endothelial cell growth, and thatendothelial cells grown on NAFION tubes by the procedures contained inthis invention resist shear forces due to fluid flow. It is generallyunderstood that a covering of only approximately 20% of the luminalsurface of a vascular graft by metabolising endothelial cells isrequired to avoid thrombogenesis and we have shown that amorphologically normal endothelial surface is achieved quickly onNAFION. This is in contrast to attempts to culture endothelial cells onTEFLON where cells remain fibroblastoid until reaching confluence andare poorly attached to the surface. Example 6 demonstrates thebiological acceptance of porous NAFION implants indicating that NAFIONmay be useful for connective tissue or soft tissue prostheses.

These results suggest that NAFION or indeed any copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomer,may be used for the in vitro attachment and growth of animal cells, andmay be incorporated into a vascular prosthesis or be a usefulalternative to commercially available materials currently used ascomponents of vascular prostheses. Preferably, the copolymer of thepresent invention may be readily cast into tubes or coated ontopre-existing tubes to serve as an effective vascular graft. Theeffectiveness of the said copolymer as a component of a vascular graftmay be further enhanced by the many apparent variations in itspreparation as discussed with reference to NAFION herein. The use of thesaid copolymer as a surface for endothelial cell attachment and growthmay be of particular value in the new technique of cell seeding ofvascular grafts and prostheses prior to implantation.

The foregoing describes only some embodiments of the present inventionand modifications obvious to those skilled in the art, can be madethereto without departing from the scope and ambit of the invention.

We claim:
 1. In a prosthesis or sponge implantable in a body, theimprovement comprising forming the surface of said prosthesis or spongefrom a neutralized copolymer of perfluoro-3,6-dioxa-4-methyl-7-octenesulphonyl fluoride and a monomer.
 2. The prosthesis or sponge of claim 1wherein the monomer is tetrafluoroethylene.
 3. The prosthesis or spongeof claim 1 further comprising a supporting material to which saidcopolymer is applied.
 4. The prosthesis or sponge of claim 3 whereinsaid supporting material is selected from the group consisting of apolymer, ceramic, metal, glass and preformed membrane.
 5. The prosthesisor sponge of claim 4 wherein said polymer is a porous polymer.
 6. Theprosthesis or sponge of claim 5 wherein said porous polymer ispolytetrafluoroethylene or expanded polytetrafluoroethylene.
 7. Theprosthesis or sponge of claim 5 wherein the porous polymer is knitted orwoven polyester.
 8. The prosthesis or sponge of claim 5 wherein theporous polymer is polyurethane.
 9. The prosthesis of sponge of claim 1in the form of a tube.
 10. The prosthesis or sponge of claim 1 whereinthe surface further comprises adsorbed adhesive proteins.
 11. Theprosthesis or sponge of claim 10 wherein said adhesive proteins arederived from serum.
 12. The prosthesis or sponge of claim 11 whereinsaid adhesive serum proteins are selected from the group consisting offibronectin, vitronectin, thrombospondin and adhesive fragments of anyof these proteins.
 13. The prosthesis or sponge of claim 1 wherein thesurface further comprises animal cells adhered thereto.
 14. A surfacefor the attachment and growth of animal cells in vivo, said surfacecomprising the neutralized form of a copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and monomerwith animal cells adhered thereto.
 15. A process for the preparation ofa surface for the attachment and growth of animal cells in vivo, saidprocess comprising applying a copolymer of perfluoro-3-,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomer to anappropriate substrate, neutralizing the resultant surface and adheringanimal cells to the surface.
 16. A process for the attachment and growthof animal cells in vivo comprising: exposing animal cells to aprosthesis or sponge having a surface formed from a neutralizedcopolymer of perfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluorideand a monomer.
 17. A process for the attachment and growth of animalcells in vitro comprising: exposing animal cells in vitro to a surfaceformed from a neutralized copolymer ofperfluoro-3,6-dioxa-4-methyl-7-octene sulphonyl fluoride and a monomer.18. The process of claim 17 which further comprises exposing saidsurface to a medium containing animal cells and adhesive proteins.